Assessment of methylation of DNA is useful in many research, diagnostic, medical, forensic, and industrial fields. Key to this assessment is converting cytosine to uracil, but not methylcytosine to thymine. One basic method for such conversion, employing sodium bisulfite, is well known. Over the years, the method has been improved in attempts to overcome disadvantages that include tedious procedures, lengthy reaction times, and DNA degradation. The currently most commonly used protocol is taught by J. Herman, Proc. Natl. Acad. Sci. 93, 9821–26 (1996), incorporated herein by reference in its entirety. This method involves denaturation, reaction with sodium bisulfite in the presence of hydroquinone, and subsequent completion of the modification by treatment with NaOH. Despite the attempts to improve the protocol, current procedures still require pre-denaturation of the genomic DNA (gDNA) to single stranded DNA (ssDNA), preparation fresh solutions of sodium bisulfite (NaHSO3), typically about 3M, and inclusion an anti-oxidant (e.g., hydroquinone). The protocol also involves long reaction times and tedious clean-up procedures.
In addition, the currently employed sodium bisulfite protocols are plagued by reports of incomplete conversion, irreproducible results, and other problems. In some cases, the reaction results in significant DNA degradation (reportedly as high as 96%), making it difficult to obtain enough sample for further analysis. See. S. J. Clark et al. Nucleic Acid Research 2001, 29 no. 13, 65.
It has been discovered that bisulfite methods that employ magnesium bisulfite, polyamine compounds, and/or quaternary amine compounds provide useful alternatives to sodium bisulfite conversion reactions. These discoveries are the subjects of co-owned applications entitled “Method And Materials For Polyamine Catalyzed Bisulfite Conversion Of Cytosine To Uracil” (U.S. application Ser. No. 60/499,113 filed Aug. 29, 2003, and also application Ser. No. 60/520,942 having the same title and filed Nov. 17, 2003), “Method And Materials For Quaternary Amine Catalyzed Bisulfite Conversion Of Cytosine To Uracil” (U.S. application Ser. No. 60/499,106 filed Aug. 29, 2003, and also application Ser. No. 60/523,054 having the same title and filed Nov. 17, 2003), and “Method and Materials for Bisulfite Conversion of Cytosine to Uracil (U.S. application Ser. No. 60/499,082 filed Aug. 29, 2003, and also application Ser. No. 60/523,056 (5180P2) having the same title and filed Nov. 17, 2003), all of which are hereby incorporated by reference in their entirety.
Two widely reported methods of purification used in connection with bisulfite conversion reactions utilize either adsorbtive resins, such as the Promega Wizard resin or similar DNA binding resins, or dialysis. Given the importance of assessment of DNA methylation, it can be seen that there is a need for improved methodology for conversion of cytosine to uracil, and for the purification of the products of such conversion. Applicants have discovered such an improved methodology which is the subject of the co-owned application entitled: “Improved Bisulfite Method” (U.S. application Ser. No. 60/498,996 filed Aug. 29, 2003, and also application Ser. No. 60/520,941 having the same title and filed Nov. 17, 2003), all of which are hereby incorporated by reference in its their entirety.